The angiotensin II type I receptor (AT1R) mediates diverse intracellular responses to angiotensin II (Ang II) in the regulation of blood pressure, hydromineral balance, cardiac hypertrophy and cell proliferation. Endocytosis of the Ang ll-bound receptor not only desensitizes AT1R signaling from the cell surface but also activates specific intracellular signaling pathways. The long-term goal of this project is to elucidate the signaling pathways that regulate AT1R subcellular trafficking. Clarifying the events that ensue will potentially suggest approaches for novel therapeutics for cardiovascular diseases related to AT1R dysfunction, and will provide new insights into the mechanism underlying receptor endocytosis in general. Although roles for many proteins in AT1R trafficking are becoming clear, less is known about the specific functions of membrane lipids. It has been recognized that interaction between proteins and membrane (particularly phospholipids) drives initiation, fission, and fusion of endocytic vesicles. However, how phospholipid metabolism is regulated during this process and, in turn, triggers signals for the different receptor endocytic steps, remains unclear. The Phospholipase D (PLD) family contains two members, PLD1 and PLD2, both of which catalyze the hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline. PLD2-generated PA has been proposed to regulate a number of endocytic and actin regulatory proteins and several types of membrane trafficking processes. The hypothesis of this proposal, based on our published work and preliminary evidence, is that PLD2-generated PA plays a central signaling role in regulation of AT1R endocytosis and signaling. In specific aim 1, we will test the hypothesis that PLD2 facilitates AT1R endocytosis by regulating spatiotemporal dynamics of phosphatidylinositol 4,5-bisphosphate {PI(4,5)P2}, which is an essential phospholipid in endocytosis. Specific aim 2 will test whether PLD2 regulates formation of clathrin-coated pits (CCPs) by determining which step in CCP formation is blocked when PLD2 is downregulated by small hairpin RNA (shRNA). We will also examine whether membrane microdomains regulated by PLD2 signaling are critical for CCP formation. Specific aim 3 will test whether differential distribution of AT1R as regulated by PLD2 determines the output of AT1R signaling. AT1R activation in different subcellular locations results in distinct signaling outputs. We will determine whether PLD2 downregulation changes the kinetic and spatial patterns of Ang II signaling, and as a result, affects cell growth. [unreadable] [unreadable]